MOLECULAR DIAGNOSTIC STUDIES:
Loss of Heterozygocity at the APC locus.
The mutational alterations being tested for in this case manifest as allelic loss, also known as loss of heterozygosity, generally regarded as a measure of tumor suppressor gene deletion. Relative intensity of polymorphic electrophoretic bands is determined from PCR amplified microsatellite tandem repeats situated in proximity to known or putative tumor suppressor genes. Each tissue target is PCR amplified using fluorescent-labeled primer and/or radionucleotide incorporation of P-33 labeled deoxynucleotide methodology. The profile of allelic loss, in each tissue target for each marker, is interpreted, compared and integrated with the histopathology.
Six different areas were selected for microssection genotyping from serial four micron histologic sections of gastrointestinal tract biopsies as follows: normal colonic epithelium (N), colonic tubulovillous adenomatous polyp (T1), colonic mucosa with early adenomatous change (T4, T5, T6), ampullary adenomatous polyp (T2) and gastric mucosa (T3). Each microdissected tissue target was assessed for allelic loss pattern with extragenic adjacent microsatellite markers for APC loss using markers MCCE10, 5q21D5S592, and 5q21D5S615. Markers, 5q21D5S592 and 5q21D5S615 were informative. No consistent pattern of allelic loss was identified. The allelic loss analysis was then extended to the APC gene itself. An informative single nucleotide sequence polymorphism in exon 15 (cytosine[c]/thymidine[t]) was evaluated in microdissected non-neoplastic and polyp tissue (Figure 7). In the non-neoplastic tissue both alleles were represented in approximately equal amounts however in the microdissected polyp tissue (T1 & T2) there was an imbalance in the representation of the individual nucletides in keeping with allelic loss (figure 7). Of note was the finding that all polyps analyzed (T1-T6) manifested the same pattern of allelic loss supporting the concept of a germline alteration in the APC gene. This provided further support for more detailed mutational screening.
Mutation Analysis of the APC gene.
A mutation scanning technique was used to determine the presence of mutations in the adenomatous polyposis coli gene (APC) gene. A PCR-based assay was used to amplify all 15 exons of the APC gene and intron/exon boundaries. The amplified products were scanned for mutations using dHPLC (Denaturating high performance liquid chromatography) (figure 8).
In this method, PCR products are denatured and allowed to rehybridize. If a mutation is present, DNA strands carrying the mutation will hybridize with strands carrying the wild type sequence, thus forming heteroduplexes. If no mutation is present, the strands will only form homoduplexes. The presence of heteroduplexes is detected by their lower melting (denaturation) point, due to the presence of a mismatch between the two strands.
PCR products (amplicons) showing variations were subsequently sequenced for mutation/polymorphism confirmation. A single base pair change of a cytosine (C) to a thymine (T) was detected at nucleotide position 481 (481 C>T), predicted to result in a change of a glutamine residue to a stop codon at codon position 161 (Q161X). The Q161X mutation identified in this patient is predicted to produce a truncated protein and is interpreted as a disease-causing mutation. These findings are consistent with the diagnosis of familial adenomatous polyposis (FAP). This nonsense mutation has been previously reported in one single other patient with an attenuated phenotype of FAP. Attenuated FAP is associated with mutations in the 5' end of the APC gene (5' to codon 158).